PURIFICATION OF THERMOTOGA-MARITIMA ENZYMES FOR THE DEGRADATION OF CELLULOSIC MATERIALS

A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed, Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography, Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, p-nitrophenyl-alpha-L-arabinofuranoside, and 4-O-methyl-glucuronosyl-xylotriose. The activities of two cellulases, one laminarinase, one xylanase, two putative beta-D-xylosidases, alpha-D-glucuronidase, and alpha-L-arabinosidase were identified, Because of their selective retardation on a Superdex gel filtration column, the two cellulases could be purified to homogeneity. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular masses of 27 and 29 kDa, respectively, were determined for cellulase I and cellulase II. Maximal activities of both enzymes were observed at 95 degrees C between pH 6.0 and 7.5. in the presence of 2.5 M NaCl the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80 degrees C. On the basis of its activity towards CMC, cellulase I was classified as endo-beta-1,4-glucanase. Cellulase II, was able to attack Avicel in addition to CMC, beta-glucan, and p-nitrophenyl-beta-D-cellobioside. It releases cellobiose and cellotriose from Avicel, The latter product is further cleaved into glucose and cellobiose. Cellulase II may therefore be classified exo-beta-1,4-glucanase.